JP5728016B2 - Process for purifying recombinant human erythropoietin (EPO), EPO thus purified and pharmaceutical composition comprising the same - Google Patents

Description

  The present invention relates to a procedure for producing erythropoietin (EPO), in particular recombinant human EPO (rhEPO) having a given composition of a highly pure form of glycoform, ie, a large amount of O-glycosylated EPO isoform. This is achieved by using a specific combination of chromatography steps.

  Erythropoietin is the main hormone that regulates the proliferation and differentiation of erythroid progenitors and the maintenance of physiological levels of circulating red blood cells. In the fetus, EPO is mainly produced in the liver, and about 90% of its production switches to the kidney after birth. If EPO levels drop due to chronic or acute renal failure, EPO must be administered externally to prevent increased anemia. Therapeutically active human erythropoietin has been available since the discovery of the EPO gene and its expression in rodent cells. Natural human erythropoietin is encoded by the gene 7q11-q22 on chromosome 7 (Law et al., Proc. Natl. Acad. Sci. USA 83 (1986), 6920-6924). Recombinant human EPO entered the market in 1989 when the US Food and Drug Administration (FDA) approved its use in the treatment of anemia associated with chronic renal failure. This is also true for other recombinant glycoproteins used as pharmaceuticals, but the glycosylation pattern of EPO has a profound effect on the bioavailability, bioactivity, and immunogenicity of glycoproteins. For production of rhEPO, Chinese hamster ovary (CHO) and baby hamster kidney (BHK-21) host cells play an important role as expression systems and are therefore well studied (Sasaki et al., J. Biol. Chem. 262 (1987), 12059-12076; Takeuchi et al., J. Biol. Chem. 263 (1988), 3657-36633; Nimtz et al., Eur. J. Biochem. 213 (1993), 39-56; Tsuda et al., Biochemistry 27. (1988), 5646-5654). It is well established that these cell lines produce glycoforms that most closely resemble human glycoforms. It is also well known that carbohydrates play an important role in glycoprotein targeting and clearance (Drickkamer et al., Annu. Rev. Cell. Biol. 9 (1003), 237-264; Helenius et al., Science 291 (2001), 2364). -2369). Recently, a new form of erythropoietin with two special N-glycosylation sites called darbepoetin alfa (ARANESP®) has been approved by the European Medicines Agency (EMEA) and the FDA.

  The human EPO gene encodes a 166 amino acid protein with a 27 amino acid signal peptide and a calculated molecular weight of 18396 daltons. Mature proteins usually have a single amino acid N-terminal deletion and are 165 amino acids long. The signal sequence directs the peptide to the intracellular compartment involved in proper glycosylation, resulting in a mature protein with three N-glycans and one O-glycan. The sugar moiety that constitutes about 40% of the total molecular weight is essential for the full biological activity of EPO in vivo. Some studies have shown that the number of terminal sialic acid residues has a positive effect on in vivo half-life, but in vitro activity, ie binding to the receptor, is highest in the non-glycosylated or partially glycosylated form (Takeuchi and Kovata, Glycobiology 1 (1991), 337-346). The degree of sialylation is directly proportional to the half-life, in which case isoforms with little sialic acid are removed from the organism much faster and thus exhibit low activity. In this context, Rush et al. (Anal. Chem. 67 (1995), 1442-1442) described their effects on O-acetylation and increased circulation time of erythropoietin sialic acid residues, which Show that high O-acetylation of sialic acid residues increases circulation time by reducing liver clearance.

  Typically, EPO preparations obtained by recombinant expression in mammalian cells, particularly CHO cells, lack E-glycans and thus have EPO isoforms that relax the possibility of more than one sialic acid residue per molecule. Includes up to 50%. Accordingly, it would be desirable to provide such defects, EPO preparations substantially free of non-O-glycosylated isoforms, and methods of providing the same.

The EPO isolation / purification methods known from the scientific and patent literature involve various chromatographic steps. The most commonly used are anion exchange chromatography (AEX) and reverse phase HPLC (RP-HPLC). Other chromatographic methods such as hydroxyapatite, hydrophobic interaction (HIC), cation exchange (CEX), affinity (ie immunoaffinity or dye ligand) and size exclusion (gel filtration) (SEC) chromatography are also used. Precipitation with several intermediate steps such as concentration, diafiltration, ultrafiltration, dialysis, ethanol, salt, etc. is also common.
EP 205564 describes the purification of EPO using a cation exchange chromatography step followed by RP-HPLC.
EP 228452 and US 4,667,016 described EPO purification with anion exchanger followed by RP chromatography and gel chromatography.
EP 428267 describes an improvement of the process according to EP 228452 which deals with the isolation of isoforms (especially acidic) by means of anion exchanger after RP chromatography.
EP 1394179 describes a purification process involving dye affinity chromatography, HIC, hydroxyapatite, RP-HPLC and anion exchangers.
EP1288878 uses a first anion exchange chromatography step and optional acid wash step as a capture step, a hydrophobic interaction chromatography, an affinity chromatography, and a second anion exchange chromatography step and an acid wash step. A purification method for highly sialylated EPO isoforms is described.
WO 99/28346 describes a high degree of EPO purification on N-acetyl-lactosamine units and / or tetra-antennary branches in carbohydrate structures. The purification process begins with cell culture supernatant and is purified by affinity chromatography, hydrophobic interaction chromatography, hydroxyapatite and reverse phase chromatography.
WO 03/045996 describes a method for recovering and purifying rhEPO from cell culture media, including, inter alia, steps of anion exchange chromatography, reverse phase chromatography, anion exchange chromatography and size exclusion chromatography. .
WO 03/080852 described a process for producing EPO, including at least dye affinity chromatography, hydrophobic chromatography and anion exchange chromatography, optionally further including gel filtration chromatography.
Miyake et al. Biol. Chem 252 (1977), 5558-5564 describes the purification of urinary EPO by a 7-step procedure including ion exchange chromatography, ethanol precipitation, gel filtration and adsorption chromatography.
Brody et al., Arch. Biochem. Biophys. 265 (1988), 329-336 used a transfected BHK cell line to purify EPO by Affi-Gel Blue chromatography, anion exchange chromatography and reverse phase chromatography.
Gokana et al. Chromatography 791 (1997), 109-118 describes the separation of recombinant human EPO isoforms by DEAE chromatography after previous purification of EPO by immunoaffinity. The separation of EPO isoforms is based on their different pi.
Goto et al., Bio / Technology 6 (1988), 67-71, describes immunoaffinity, gel chromatography and purification of EPO with hydroxyapatite.
Hokke et al., Eur. J. et al. Biochem. 228 (1995), 981-1008 describes glycan analysis of EPO, including PNGase treatment for individual analysis of N-glycans and O-glycans.
Kishino and Miyazaki, J. et al. Chromatography 699 (1997), 371-381 outlines a method for glycoprotein analysis that includes purifying EPO from urine using terminal RP chromatography.
Nimtz et al., Eur. J. et al. Biochem. 213 (1993), 39-56 describe an analysis of EPO glycosylation, including PNGase F digestion, which is expressed in baby hamster kidney (BHK) cells and 60% of recombinant human EPO obtained from BHK cells. Only O-glycosylated.
Quelle et al., Blood 74 (1989), 652-657 describes the purification of EPO from insect cells, where the purification process includes anion exchange chromatography and RP-HPLC followed by lectin affinity chromatography.
Sasaki et al. Biol. Chem. 262 (1987), 12059-12076 and Sasaki et al., Biochemistry 27 (1988) 8618-8626 describe the carbohydrate structure analysis of recombinant human EPO expressed in and obtained from Chinese hamster ovary (CHO) cells. ing. EPO was purified by RP-HPLC among other methods.
Skibell et al., Blood 98 (2001), 3626-3634, describes an investigation of the glycan structure of human serum-derived EPO compared to recombinant EPO, and that O-glycosylation also occurs in EPO circulating in the blood Indicates.
Sytkowski and Donahue J .; Biol. Chem. 262 (1987), 1161-1166 determined the EPO receptor binding site determined with the aid of a monoclonal antibody (mAb) (indirectly by neutralization). From Table II, it can be seen that mAbs against EPO amino acid sequences 111-129 show the strongest neutralizing effect, which means that O-glycans located at Ser126 may be involved in receptor binding.

  Thus, although the importance of glycosylation of EPO is known, all of the above documents are (i) substantially free of non-O-glycosylated isoforms, (ii) purified to pharmaceutical grade, (iii) viruses And (iv) does not disclose a method for providing EPO preparations that can be manufactured on a large scale. This technical problem is solved by the embodiments characterized in the claims and described in more detail below.

  The present invention is a method for recovering and purifying human erythropoietin (rhEPO) from cell cultures derived from recombinant cells, comprising a reverse phase (RP) chromatography step, preferably RP-HPLC and subsequent cation exchange ( A method comprising a CEX) chromatography step is provided, wherein the RP-HPLC step is preferably performed after the anion exchange (AEX) chromatography step.

  Recombinant human erythropoietin (rhEPO) purified according to the method of the present invention has been subjected to several analytical procedures to characterize the N- and O-glycosylation status of EPO. As described in the background section, EPO has one O-glycosylation site at S126 and three N-glycosylation sites at N24, N38, and N83. The overall glycosylation pattern of the EPO preparation of the present invention was found to be quite similar to that found in international BRP standards and reference products. However, for O-linked oligosaccharides, it was observed that the relative amount, if any, of the non-glycosylated form of EPO compared to the BRP standard. This may mean that the EPO preparation of the present invention is improved compared to other commercial products.

  While not intending to be bound by theory, it is believed that substantially selective separation of O-deglycosylated (Des-O) EPO functions in HPLC due to the fact that all O-glycans are loaded. . In the case of N-glycans, the difference, if any, is not so noticeable. Here, only accidental antennas are missing, but all glycans are almost missing. Isoforms lacking N-glycans (there are no 4 sialic acids per glycan) have already been strongly excluded in the anion exchange chromatography (AEX) step. In the absence of an O chain, a hydrophobic patch (approximately aa121-130) that is usually protected by a sugar chain is exposed. In fact, a closer look at the O-glycosylation site shows a dense accumulation of hydrophobic amino acids around serine 126. Four alanines directly surround serine 126 and three prolines (121, 122, 129) define a pronounced hydrophobic region. In addition, leucine 130 is also hydrophobic and contributes to this effect. The additional hydrophobic site in EPO enhances the overall binding strength of the HPLC column to the C4 matrix. Thus, the Des-O form elutes at a later time.

In a particularly preferred embodiment of the present invention, a 5-column chromatography process for purifying EPO in a pure form suitable as a drug substance is provided, the process comprising:
(A) Dye affinity chromatography to capture, concentrate and provide a significant reduction in potential contaminants for EPO-containing solutions;
(B) Anion exchange chromatography for concentration of acidic isoforms of EPO, further removal of contaminants (eg, DNA, HCP), and removal of any dye ligand that may have leached from the first column;
(C) RP-HPLC under conditions to remove EPO molecules that are not glycosylated at Ser126 residue and to remove contaminants;
(D) cation-exchange chromatography to remove RP-HPLC solvents and aggregated species, exchange buffers, and concentrate EPO fractions; and (e) possible aggregates and other Includes size exclusion chromatography as a final chromatography step used to remove contaminants.

  In addition, two specific virus removal / inactivation steps are included in the EPO manufacturing process. First, after the RP-HPLC step, the eluate is kept at 22 ± 3 ° C. for 60-18 minutes in acidic acetonitrile: water + 0.1% (v / v) TFA mixture. Second, the nanofiltration step is performed after the final chromatography step, increasing the virus safety of the drug substance. In addition, anion exchange chromatography has proven to be an effective step for virus removal. The resulting EPO drug substance can be dispensed, for example using a peristaltic pump, into a bulk drug substance storage container with a volume of 250 ml or 30 ml and stored at -70 ° C or lower.

  Thus, the method of the present invention provides a profile-dependent production of EPO and a high quality, very uniform product. Furthermore, the present invention allows for large scale preparation of bioactive EPO with high purity and the desired O-glycosylated EPO isoform profile.

  EPO purity is high by reaching a purity of at least 99% of the total protein, and advantageously greater than 99.9% of the total protein, as measured by HPLC and gel electrophoresis. Furthermore, by reducing the risk of infection, the risk of contamination by viruses etc. and thus the clinical safety of the product is improved. In this regard, the advantageous side effects of using RP-HPLC and organic solvents to elute and store EPO samples during the next step in the purification process are, for example, hydrophobic interactions or ions It is in the inactivation of viruses that can remain viable in other solvents used in other methods such as exchange chromatography.

  In this context, those skilled in the art will appreciate that one essential feature of the present invention consists of RP-HPLC performance and subsequent CEX chromatography steps, while any of the other chromatography steps can be modified. It will be understood that it can be done or omitted altogether. This also applies to the AEX step, which is preferably incorporated into the method of the invention, but can be replaced by a different purification step, and further depending on the applied EPO sample and / or O -If it is desired to provide EPO preparations that are glycosylated while being heterologous or hyposialylated, for example, they can be omitted

  The fact that the O-glycosylation of EPO isolated according to the method of the invention was more complete than the BRP standard indicates that the half-life of the EPO preparation can be greatly improved. Thus, the in vivo half-life of the EPO preparation of the present invention is expected to be at least similar, if not improved, compared to the recombinant EPO product that was previously commercially available. Is wise. This also means that the pharmacokinetic and pharmacodynamic properties would otherwise be similar to market products. The EPO obtained according to the method of the invention is therefore particularly suitable for use in human medicine.

A patent or application may contain one or more drawings made in color and / or one or more photographs. Copies of this patent or patent application publication with color drawings and / or photographs may be provided by the European Patent Office or US Patent and Trademark Office upon request and payment of the necessary fee.
Scheme of purification procedure for isolation of specific mixtures of highly sialylated and O-glycosylated EPO isoforms. The importance and intended purpose of the individual purification steps are further explained in the specification. Photo of Coomassie stained 12.5% SDS-PAGE gel of HPLC fraction after digestion with PNGase. 5 μg of protein was loaded per slot. MW: molecular weight marker; BRP: BRP standard batch 1 (= biological reference preparation = mixture of epoetin alfa and beta); 1 = EPO sample after anion exchange chromatography (AEX pool) and before subjecting to RP-HPLC step 2-10 = A sample of EPO eluate fraction obtained after subjecting the AEX pool to RP-HPLC and applying a linear gradient of solvent as described in the examples; see also Table 6. Each EPO batch is shown after digestion with polypeptide N-glycosidase (PNGase), which removes all N-glycans, but if present, leaves bound O-glycans. The lower thin band is the non-O-glycosylated (Des-O) form. The enzyme itself may be detected in the gel as a trace band in the middle of the gel. The left lane shows the BRP standard, the next lane shows the column application (including the Des-O form like the BRP standard), followed by 9 fractions of gradient elution. The first five fractions clearly have no Des-O form and the Des-O form is mainly present in the last 3-4 fractions, which indicates that the Des-O isoform is bound to the chromatographic material. And is eluted from the column at a later time. Pool criteria adjust the residual rate of Des-O isoform. Photo of a Coomassie stained 12.5% SDS-PAGE gel. MW: molecular weight marker; 1-4 = EPO preparation obtained by the method described in the examples; 5 + 6 = BRP standard batch 1 (= biological reference preparation = mixture of epoetin alfa and beta); 7 = blank. Each EPO batch is shown before digestion with PNGase (1, 3, 5) and after digestion (2, 4, 6). The enzyme itself can also be detected as a trace band in the gel. The BRP standard shows a double band after digestion. The lower narrow band is the non-O-glycosylated (Des-O) form. The EPO preparation obtained according to the method of the present invention clearly shows no lower band, confirming that the Des-O form was removed during the HPLC step.

  The present invention provides an improved EPO preparation isolation and purification process that contains few, if any, non-O-glycosylated isoforms and is substantially free of aggregates. The purpose is to determine the content of non-O-glycosylated EPO isoforms by applying a reverse phase (RP) -high pressure liquid chromatography (HPLC) followed by a cation exchange chromatography (CEX) step under appropriate conditions. This is achieved by reducing. More particularly, the present invention relates to a method for purifying glycosylated erythropoietin (EPO) isoforms from complex protein mixtures, which comprises an anion exchange (AEX) chromatography step and a cation exchange (CEX) chromatography step. Which are separated by a reverse phase (RP) chromatography step.

  The term “isoform” as used herein has the same amino acid sequence but a different isoelectric point or for example capillary zone electrophoresis (eg, as revealed by fractional electrophoresis (IEF) gels). Refers to a glycoprotein preparation / fraction containing glycoproteins with different charge numbers as revealed by CZE). These differences reflect heterogeneity in the glycosylation pattern. Individual EPO molecules can differ with respect to the extent, complexity, nature, branching, and order of the attached glycosyl, sialyl, and acetyl groups. Even charged inorganic groups such as phosphates and sulfates can contribute to the properties of certain isoforms. Thus, glycoprotein isoforms according to the present invention are defined by their different isoelectric points and their same amino acid sequence, and thus each isoform actually contains multiple different EPO molecules in a strict chemical sense.

  According to the method of the present invention, anion exchange chromatography is preferably used to select EPO isoforms, particularly highly sialylated acidic EPO isoforms; see also FIG. According to the present invention, “acidic isoforms” of EPO include isoforms that have a high or high content of glycosyl groups, preferably sialyl groups, and thus appear at low (acid) pI. Recombinant human EPO exhibits a broad isoelectric (pI) isoform profile of up to 14 different isoforms over the range of pI3-9 when analyzed from cell culture supernatants by IEF (Gokana et al., J. Chromatogr 791 (1997), 109-118). EPO isoforms arise mainly due to different glycosyl contents with different numbers of negatively charged terminal sialic acid residues. It is known that EPO forms with more sialic acid residues and thus with a more acidic pI are of higher biological activity and therapeutic value. This is because the terminal sialic acid residue on the glycostructure prevents EPO from being rapidly cleared in vivo by the asialo receptor pathway.

  The anion exchange chromatography step can be performed using a diethylaminoethyl group (DEAE), a quaternary aminoethyl group (QAE), a quaternary ammonium group (Q), a dimethylaminoethyl group (DMAE) and / or a trimethylaminoethyl group (TMAE). Can be carried out using an anion exchange resin or membrane containing as a functional group. Examples of anion exchange materials are Dowex® 1, AG® (eg, types 1, 2, 4), Bio-Rex® 5, DEAE Bio-Gel 1 available from Dow chemical company. , Macro-Prep® DEAE (all available from BioRad Laboratories), Eicrom Technologies Inc. Anion exchange resin type 1 available from, Source Q, ANX Sepharose 4, DEAE Sepharose (eg, type CL-6B, FF), Q Sepharose, Capto Q, Capto S (all available from GE Healthcare), AX-300 (Available from PerkinElmer), Asahipak ES-502C, AXpak WA (eg, type 624, G), IEC DEAE (all available from Shoko America Inc.), Amberlite® IRA-96, Toyopearl trademark DEAE, TSKgel DEAE (all available from Tosoh Bioscience GmbH), Mustang Q (Pall Corpora) It is available) from the ion. In a preferred embodiment of the method of the invention, an anion exchange chromatography step is performed using Q-Sepharose; see also the examples. As described, the depletion of the low sialylated basic EPO isoform and the selection of the highly sialylated acidic EPO isoform, respectively, preferably in a buffer (pH about 7.0) containing 20 mM Tris-HCl, Run with a linear salt gradient of 0-200 mM NaCl.

Furthermore, according to the method of the present invention, reverse phase chromatography steps are used to select O-glycosylated EPO isoforms. As shown in the Examples, such EPO isoforms can be eluted with a linear gradient of organic solvent (preferably 0-70% acetonitrile in water (containing about 0.1% TFA)). In addition or alternatively, isocratic elution of EPO with a solvent containing acetonitrile and about 0.1% TFA in water is used. Means and methods for performing reverse phase (RP) chromatography are well known to those skilled in the art; see also the prior art described in the background section above. Preferably, the RP chromatography step comprises reverse phase high performance liquid chromatography (RP-HPLC). Typically, RP-HPLC is carried out using a resin containing methyl, butyl, phenyl, propyl and / or octyl groups as functional groups. In a preferred embodiment of the method of the invention, RP-HPLC is performed using commercially available C4 reverse phase chromatography material. Examples of reversed phase materials are: Vydac 214TPB1015, C4 (available from Grace Davison); Daisopak SP-300-15-C4-BIO (available from DAISO Fine Chem. GmbH); YMC Gel Butyl Sharsch C4, 15μ, A YMC Europe GmbH); Jupiter 15μ, C4, 300A (available from Phenomenex).
Most preferably, Vydac C4 (Vydac) composed of silica gel particles whose surface has C4-alkyl chains is used. Separation of EPO from proteinaceous impurities is based on differences in the strength of hydrophobic interactions. Elution is performed with an acetonitrile gradient in water in the presence of dilute trifluoroacetic acid. Usually, part of the “EPO peak” has to be cut off by fractionation. For example, in eluate fractions containing many about 9 or 10 EPO, the last 1-4 samples that typically may constitute 40% of the total EPO peak must be discarded, while the remaining eluate The pool is further purified in the next step; see also FIG.

  According to the present invention, preparative RP-HPLC is usually carried out with high pressures> 10 bar (on a preparative scale up to 30-40 bar) and small beads (5-10 μm), usually silica gel, which is better Resolution. Preferably, the pH of the solvent acidified with TFA is about 2. Application of AEX eluent at low acetonitrile concentrations (eg, 10% or pure water) and increasing acetonitrile concentration gradients will elute EPO isoforms.

  In this context, RP-HPLC, when used according to the method of the present invention, is a conventional RP chromatography (RPC) performed at low pressure (also called medium pressure method, <10 bar, usually 3-5 bar). And substantially different. For example, RPC is typically performed with 15μ beads or 30μ beads as described in International Application No. WO 03/045996 (RP-Source 30). In addition, organic solvents can be used as in HPLC, but the RPC step in WO 03/045996 begins with ammonium sulfate precipitation with 0.24 M ammonium sulfate in the sample, which is preferred for HPLC. Not very well, but very suitable for HIC (Hydrophobic Interaction Chromatography) where a salt gradient that typically decreases due to elution is applied. Thus, RPC as described in International Application No. WO 03/045996 is carried out in a sufficient aqueous solvent system, ie Tris / HCl buffer (pH 7). This is because, until now, no suitable method has been described to avoid rapid separation of organic solvents and formation of aggregates / dragging within EPO preparations. However, the implementation of HPLC steps using organic solvents is at the expense of less efficient separation of EPO isoforms due to, for example, the larger bead particles used in RPC and less stringent separation conditions. In any case, the separation performance of RP-HPLC is superior to RPC.

  A further important aspect of the effectiveness of the purification scheme is that cation exchange chromatography (CEX) is performed in the third step, immediately after HPLC. This step is new with respect to the application scheme. After RP chromatography, EPO must be subjected to buffer exchange immediately. This is because it is not stable in acidic acetonitrile, ie, aggregates are gradually formed with increasing time and temperature. Furthermore, for subsequent gel chromatography used as the final polishing step, the sample volume must be very small. That is, the EPO concentration in the RP pool must be increased strongly. On the other hand, the RP-HPLC pool has a fairly large volume due to the relatively flat gradient required for separation of the Des-O form. Instead of using a standard ultrafiltration / diafiltration step, which is a common but rather time consuming method, a CEX chromatography step with a material selected according to the present invention (Macroprep S) was developed. It was. This CEX chromatography allows for particularly high flow rates and is tolerant to acidic acetonitrile at high concentrations. Due to the fact that it is positively charged in an acidic environment, EPO is a strong binder, eluting at a high concentration in the desired buffer, with a very steep slope, in a small volume. Surprisingly, CEX chromatography also leads to separation of EPO aggregates that are inevitably formed in acetonitrile. This is because it was found that the EPO aggregates were not eluted but remained on the column and then eluted into the regenerated product with the CIP solution. Thus, according to the present invention, the cation exchange chromatography step is used after RP chromatography, especially RP-HPLC for buffer exchange, concentration of EPO and removal of EPO aggregates; see also FIG.

  Different types of cation exchange materials are also available from a number of companies with different names. For example, Bio-Rex (registered trademark) (for example, type 70), Chelex (registered trademark) (for example, type 100), Macro-Prep (registered trademark) (for example, type CM, High S, 25S), AG (registered) Trademarks) (eg, type 50W, MP) (all available from BioRad Laboratories); WCX2 (available from Ciphergen), Dowex® MAC-3 (available from Dow chemical), Mustang C and Mustang S ( Available from Pall Corporation), Cellulose CM (eg, type 23, 52), hyper-D, PartiSphere (available from Whatman plc.), Amberlite® I RC (eg type 76, 747, 748), Amberlite® GT73, Toyopearl® (eg type SP, CM, 650M) (all available from Tosoh Bioscience GmbH), CM1500 and CM3000 (BioChrom Labs) SP-Sepharose ™, CM-Sepharose ™ (available from GE Healthcare), Porous resin (available from PerSeptive Biosystems), Asahipake ES (eg, type 502C), CXpak P, IEC (Eg type 825, 2825, 5025, LG), IEC SP (eg type 420N, 825), IEC QA (eg , Type LG, 825) (available from Shoko America Inc.), available from 50W cation exchange resin (Eichrom Technologies Inc.). Preferably, the cation exchange material is a strong cation exchange material such as Macro-Prep® High S or 25S, MacroCap SP, Toyopearl® SP650M, Source S, SP Sepharose, or POLYCAT A. In one embodiment, the cation exchange material is a sulfopropyl cation exchange material. In a preferred embodiment of the method of the invention, the cation exchange chromatography step is carried out with Macro-Prep High S; see also the examples.

In a preferred embodiment of the method of the invention, an affinity chromatography step is performed as a capture step before the chromatography step. Usually, the affinity chromatography step is carried out with a dye chromatography resin, such as the commercially available Blue-Sepharose; see also the examples. Preferably, the chromatography steps in the method of the invention are performed in the following order:
(A) an affinity chromatography step as a capture step;
(B) anion exchange chromatography step;
(C) reverse phase (RP) chromatography step; and (d) cation exchange chromatography step; see also FIG. 1 and the Examples.
In the first step, dye chromatography removes contamination mainly by proteases. Blue triazine dyes such as Cibachron® blue are preferably used as the dye. Other triazine dyes are also suitable. The support material for the dye chromatography is not critical, but polysaccharide-based support materials such as Sepharose, preferably Sepharose 6 Fast Flow are preferably used. The enrichment of highly sialylated and O-glycosylated EPO isoforms according to the present invention is performed in a subsequent chromatography step; see also FIG.

  In a preferred embodiment of the method of the invention, elution of EPO in one or more chromatography steps is performed by step or gradient elution. The terms “step elution” and “step elution method” are used interchangeably in this application to determine the concentration of a substance that causes elution, ie dissolution of the bound compound from the material, at a time, ie one value / level. Means to raise or lower from one to the next value / level. In this “step elution”, one or more conditions, such as pH, ionic strength, salt concentration, organic compound concentration, and / or chromatographic flow, are simultaneously measured from a first value, eg, starting value, to a second To a final value, for example. This means that the condition is changed gradually, i.e. stepwise, for changes that are definitely linear or non-linear. In the “step elution method”, new fractions are collected after each increase in ionic strength or organic solvent content. This fraction contains compounds recovered from the ion exchange material, each with a corresponding increase in ionic strength and hydrophobicity. After each increase, the conditions are maintained until the next step in the elution method is performed. Typically, in large scale production, gradient elution is changed to step or isocratic elution whenever possible.

  In this regard, any of the chromatographic steps performed according to the method of the present invention can be performed in either a gradient or isocratic manner; for reviews see, for example, Schellinger and Carr, J. et al. Chromatography. 1109 (2006), 253-266. In particular, it is envisaged to change the linear gradient to isocratic elution for RP-HPLC and anion chromatography (AEX) steps. Thus, in one embodiment of the method of the present invention, the RP and / or AEX step is performed by isocratic elution.

  In further chromatographic steps of the method of the present invention, potential dimers, high aggregates and unwanted small molecules such as process related impurities are removed and, where appropriate, buffer exchange of the final formulation is also performed. Polish the EPO preparation obtained from cation exchange chromatography (CEX) by size exclusion chromatography (SEC). Typically, the size exclusion chromatography step is performed using a gel filtration medium selected from the group of Superdex, Sephacryl, Sephadex, Sepharose, Fractogel, Toyopearl and Bio-Gel. Preferably, the size exclusion chromatography step is performed on a commercially available Superdex-S200; see also the examples.

  In order to achieve a high product concentration of the EPO preparation obtained after CEX chromatography, the eluate is preferably further concentrated prior to gel filtration (SEC). This is usually performed by an ultrafiltration step using a 5-10 kDa UF membrane, resulting in an approximately 10-fold concentrated UF residue containing about 5-20 mg EPO per ml, resulting in a SEC pool; See also Examples.

  In order to remove potential virus contamination, an additional dead-end virus-filtration step is performed with the method of the present invention. This filtration is performed with a special membrane designed to remove particles as small as 15 nm, such as Planova 15N (Asahi). Another dead-end nanofiltration unit is a PALL Ultimate VF Grade DV20 or Millipore Virsolve NFP cartridge or capsule. There are few other means of virus removal or inactivation, particularly for small envelopeless viruses (eg, parvovirus). The sterilized SEC pool is passed through a dead end filter with a suitable membrane and the filtrate corresponds to the final bulk drug substance. Alternatively, nanofiltration can be inserted between UF concentration and size exclusion chromatography. Thus, the method of the invention may comprise an ultrafiltration step and optionally a nanofiltration step before the one or more chromatography steps (the latter is preferably as the final step); see also FIG.

In a particularly preferred embodiment, the method of the invention comprises the following step (a) affinity chromatography step as capture step;
(B) anion exchange chromatography step;
(C) reverse phase (RP) chromatography step;
(D) a cation exchange chromatography step;
(E) size exclusion chromatography step;
(F) a nanofiltration step; and (g) an ultrafiltration step prior to steps (a), (b) and / or (e).

  The content of the different fractions can be measured by SDS-PAGE as an in-process control and the selected fractions are combined or discarded as necessary; for example, for controlling EPO elution from the RP-HPLC step See FIG.

  In a preferred embodiment, the EPO purified according to the method of the invention is human recombinant EPO. Thus, an EPO molecule is preferably provided by inducing expression of an EPO-encoding gene in a host cell. A “host cell” is understood as an animal or human cell whose genome contains an active EPO gene, which is transcribed and translated during the cultivation of the cells in serum-free medium. The EPO gene can be introduced into this host cell as a foreign gene, preferably with regulatory elements (see, for example, European patent applications EP-B0148605 and EP-B0209) or active in the host cell. May already exist as an endogenous gene or become activated as an endogenous inactive gene. Activation of such endogenous genes can be achieved, for example, by specifically introducing regulatory elements into the genome by homologous recombination. International applications WO 91/09955 and WO 93/09222 describe examples of such methods.

  Mammalian cells are usually used as host cells. When introducing an exogenous human EPO gene, for example, CHO or BHK cells can be used as host cells. When using the endogenous EPO gene for expression, it is advantageous to use human cells such as kidney, liver or lymphocytes. Preferably, the EPO is human recombinant EPO produced in CHO cells. Recombinant production of EPO in CHO is usually performed by adding fetal calf serum and optionally bovine insulin to the medium. As a result, EPO preparations produced in this way carry a potential risk of infection with viruses or TSE inducers. This is because at least trace amounts of such animal-derived agents can be included even after purification. It is known that serum-free culture of recombinant CHO cells containing the EPO gene can be carried out using state-of-the-art methods; , As well as in general form, Kawamoto et al., Analytical Biochem. 130 (1983) 445-453, Kowar and Franek, Methods in Enzymology 421 (1986), 277-292, Bavister, Expology 271 (1981), 45-51, European Patent Application Nos. EP0248656, EP0481772 No., EP 0 343 635 and International Application No. WO 88/00967, the disclosure of which is incorporated herein by reference. EPO derivatives and fragments that have similar activity and are produced after culturing EPO-producing host cells can also be produced in pure form according to the process according to the invention. The DNA and protein sequences of human EPO are described, for example, in European Patent Applications EP 0205564 and EP 0209539.

  In order to produce EPO, host cells containing the EPO gene can be adapted to a medium free of proteins of natural origin by passage in low volume cultures. The adapted cells are optionally cryopreserved, optionally obtained from an established cell bank and grown in serum-free medium as described, for example, in European Patent Application No. EP 1394179. For purification, the cell-free culture supernatant of the host cells is preferably isolated and subjected to the purification process according to the invention after filtration. Before carrying out the purification process, further filtration can be carried out if necessary to carry out turbidity or debris separation and / or concentration by ultrafiltration.

In a further aspect, the present invention relates to a preparation of glycosylated EPO isoforms purified by the method of the invention as described above, preferably carried out as described in the examples. Typically, EPO is human recombinant EPO. Advantageously, the EPO preparation of the invention is substantially free of non-O-glycosylated EPO isoforms. The term “substantially free of non-O-glycosylated EPO isoform” means that an EPO preparation of the invention typically has less than 10% non-O-glycosylated EPO isoform, preferably less than 5%. And advantageously comprises less than 1% non-O-glycosylated EPO isoforms. In other words, EPO preparations of the present invention typically comprise at least 90% O-glycosylated EPO isoform, preferably at least 95%, and most preferably at least 99% O-glycosylated EPO isoform. This allows the EPO preparations of the present invention to be distinguished from EPO preparations purified according to methods known in the art, for example, by SDS page as shown in FIGS. The main analytical techniques for characterizing the glycosylation state are MALDI / TOF-MS and HPAEC-PAD. Other techniques for characterizing the samples included SDS-PAGE, IEF, UV, CD, fluorescence spectroscopy and NMR. For example, quantification of O-glycosylated preparations of EPO is described in Eur. Pharm. Can be performed by MALDI / TOF-MS analysis of tryptic peptides of EPO molecules after separation of the peptide mixture through the RP-HPLC C ₄ -phase according to The non-glycosylated tryptic peptide containing the Ser-126 moiety has a mass of 1466.6, while the corresponding peptide with the GalNAc moiety (Hex-NAc) has a mass increase of 203 (m / z = 1669). The Gal-GalNAc (Hex-NAc-Hex) derivative should have an increased mass of 365 corresponding to a mass of m / z = 1830. Corresponding experiments performed within the scope of the present invention using MALDI / TOF-MS analysis showed that substantially compared to about 15% non-O-glycosylated isoforms present in standard EPO preparations. The SDS-PAGE pattern of the EPO preparation after release of N-glycans by polypeptide-N glycosidase (PNGase) was confirmed in that only the O-glycosylated form of the EPO protein could be detected.

  In this context, the minimum value of EPO specific activity is typically 100,000 IU / mg (glycoprotein). In one embodiment, the EPO preparations of the invention have an activity of> 110,000 IU / mg, which can be achieved by concentration of highly sialylated EPO isoforms.

  Thus, the EPO preparation of the present invention is particularly suitable for therapeutic applications. The invention therefore also relates to a pharmaceutical composition comprising an EPO preparation of the invention as defined above. In this context, the invention also relates to a process for producing a pharmaceutical composition, which process prepares, isolates and thus prepares EPO in the form of a glycoisoform mixture as defined above. Providing a mixture of the isolated EPO with a pharmaceutically acceptable carrier. In one embodiment, the present invention comprises tris- (hydroxymethyl) -aminomethane as a stabilizer (and the formulation does not contain amino acids or human serum albumin), most preferably sodium phosphate buffer as a pH buffer, Relates to a stable pharmaceutical formulation comprising tris- (hydroxymethyl) -aminomethane as stabilizer in an amount of 10-200 mM and / or NaCl in an amount of 20-150 mM and a pharmaceutical amount of purified EPO . For a further embodiment of the EPO formulation of the present invention, see European Patent Application No. EP 1537876, the disclosure of which is incorporated herein by reference. The pharmaceutical composition of the present invention is characterized as being at least sterile and nonpyrogenic. As used herein, “pharmaceutical composition” includes formulations for human and veterinary use. Methods for preparing the pharmaceutical compositions of the present invention are within the skill of those in the art, see for example Remington's Pharmaceutical Science, 17th ed. , Mack Publishing Company, Easton, Pa. (1985) and the latest edition of Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, the entire disclosure of which is incorporated herein by reference. ).

  These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature on any one of the materials, methods, uses and compounds used in accordance with the present invention can be retrieved from public libraries and databases. For example, a public database “Medline” can be used, which is hosted by the National Center for Biotechnology Information and / or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses such as those of the European Institute for Bioinformatics (EBI) (part of the European Institute for Molecular Biology (EMBL)) are known to those skilled in the art and can also be obtained using internet search engines. it can. An overview of patent information in biotechnology and a search for relevant patent information sources useful for retrospective searching and current awareness is described in Berks, TIBTECH 12 (1994), 352-364.

  The above disclosure generally describes the present invention. Unless otherwise stated, the terms used herein are defined as described in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, 1997, 2000 revision, 2003 reprint, ISBN0198650632). Is. Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references cited throughout this application, issued patents, published patent applications, and manufacturer specifications, instructions, etc.) are hereby expressly incorporated by reference. Incorporated in; but does not imply that the cited document is actually prior art of the present invention.

  A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. In particular, the examples relate to preferred embodiments of the invention.

Unless defined otherwise, all technical and scientific terms used herein refer to the technical field (eg, cell culture, molecular genetics, nucleic acid chemistry, hybridization technology, protein chemistry and biochemistry). Has the same meaning as commonly understood by those having ordinary skill in the art. Molecule, the genetic and biochemical methods (see generally, Sambrook et al, Molecular Cloning:.. A Laboratory Manual, 2 nd ed (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y and Ausubel et al, Short ^{Protocols in Molecular Biology (1999) 4} th Ed, John Wiley & Sons, Inc. and the title Current Protocols in Molecular see (incorporated herein by reference) full of Biology) and standard chemical methods Techniques are used.

Medium containing starting EPO protein is produced by large-scale perfusion culture of a transformed Chinese hamster ovary cell line (CHO dhfr ⁻ ) containing an amplified human EPO gene, as described in the art. See, for example, the aforementioned literature. The concentration of EPO protein in the medium is related to cell growth. In practice, the product expression level (mg / volume) is limited by the maximum number of cells achieved. Therefore, production is maximized using a continuous perfusion culture system that allows high density cell culture over long culture periods. Perfusion is initiated at a predetermined cell density, and the perfusion rate is maintained within a predetermined range by manually adjusting the rate relative to the number of cells. Cell retention is achieved by standard procedures such as acoustic precipitation tanks, filtration or centrifugation. The perfusate harvest is collected in several portions at the cooled temperature. The overall method typically consists of several weeks of perfusion culture, yielding 5-10 harvests. Residual cells and debris in the perfusate are removed from the perfusate harvest by depth filtration, and the protein is concentrated by tangential flow ultrafiltration, preferably with a molecular weight cutoff of 30 kDa. The concentrate is divided into aliquots and stored at temperatures below −70 ° C. until the entire harvest is collected. The concentrated harvest is removed from the freezer and placed in a freeze-thaw unit. The pooled and thawed harvest is clarified by filtering through a 0.45 μm depth filter. All downstream purification steps after thawing are performed at ambient temperature (17-25 ° C.).
Buffer and Solution Composition Buffer solutions for each purification step are listed in Table 1 below.

緩衝液組成

Buffer composition

表１（続き）：緩衝液組成

Table 1 (continued): Buffer composition

  Each pooled concentrate is purified using five subsequent column chromatography processes to obtain a batch of therapeutic grade highly sialylated and O-glycosylated EPO; see also FIG.

Capturing EPO and reducing potential contaminants by affinity chromatography using Blue Sepharose 6FF Blue Sepharose 6FF is an agarose resin covalently linked to the dye Cibacron Blue in the presence of contaminants in the culture harvest. Used to combine EPO and priority. The product stream is first purified by affinity chromatography as defined in Table 2.

Ｂｌｕｅ Ｓｅｐｈａｒｏｓｅクロマトグラフィーの性能に関するパラメータ

Parameters for the performance of Blue Sepharose chromatography

  The column is packed with Blue Sepharose 6FF resin. After packing, the column is tested for theoretical plates and asymmetry factors. The column is sterilized with 0.5 M NaOH for 1 hour and then rinsed with water for injection (WFI). Prior to loading, the column was washed with 20 mM Tris. Equilibrated with HCl, 1.5 M NaCl, pH 7.5, followed by 20 mM Tris. Equilibrate with HCl, pH 7.5. The sample is loaded onto the column and the column is loaded with 20 mM Tris. Wash with HCl, pH 7.5. 20 mM Tris. The sample is eluted using HCl, 1.5 M NaCl, pH 7.5. Sample collection begins after the minimum OD between the pre-peak and main peak is 0.15 or higher. When the OD becomes 0.15 or less, the collection is stopped. Collect the eluate in a sterile disposable bag.

  After elution, the column is rinsed with WFI and then stripped by washing with 5M urea. After further washing with WFI, the column is sterilized with 0.5 M NaOH. The column is rinsed with WFI and stored in 0.01 M NaOH.

  Further information on chromatography resins is provided in the manufacturer's Regulatory Support File (Representative GE Healthcare Blue Sepharose Regulatory Support File).

Concentration of EPO by diafiltration The eluate is concentrated by ultrafiltration and diafiltered in a tangential flow filtration unit using a 10 kDa cutoff membrane; see Table 3.

ダイアフィルトレーションの性能のパラメータ

Diafiltration performance parameters

  After performing the normalized water permeability test (NWP), the filter unit is used. The filter unit is sterilized with 1M NaOH for at least 1 hour and then washed with WFI. After washing, the filter unit is equilibrated with 20 mM Tris-HCl, pH 7.0. The sample is then loaded and filtered with a transmembrane pressure of 1 bar or less. Diafiltration stops when the permeate conductivity is less than 2.5 mS / cm.

Concentration of acidic isoforms of EPO and further removal of contaminants by anion exchange chromatography on Q-Sepharose HP Anion exchange chromatography on Q-Sepharose HP resin was performed to concentrate acidic isoforms of EPO, contaminants (eg, Used for further removal of DNA, HCP) and any dye ligand that may be leached from the first column. In addition, anion exchange chromatography is an effective step for exogenous virus removal. Therefore, the diafiltration solution is treated by anion exchange chromatography as specified in Table 4, followed by 0.2 μm filtration of the Q eluate fraction and fraction pool.

性能アニオン交換クロマトグラフィーのパラメータ

Performance anion exchange chromatography parameters

  The column is packed with Q-Sepharose HP resin. After packing, the column is tested for theoretical plates and asymmetry factors. The column is rinsed with WFI and then sterilized with 1M NaOH for 1 hour. After sterilization, the column is rinsed with WFI. Prior to loading, the column was washed with 20 mM Tris. Equilibrated with HCl, 1.0 M NaCl, pH 7.0, followed by 20 mM Tris. HCl, pH 7.0. Equilibrate with. The sample is loaded onto the column and the column is loaded with 20 mM Tris. Wash with HCl, pH 7.0. 20 mM Tris. The sample is eluted with a linear gradient using HCl, 0.5 M NaCl, pH 7.0. EPO is collected as a fraction with a linear gradient. The core fraction starts at about 85% to 95% of the negative gradient (UV) maximum peak and stops when the UV value decreases to about 25% of the maximum peak.

  Each anion exchange chromatography eluate fraction is filtered using a 0.2 μm filter unit. The fractions are kept at 22 ± 3 ° C. and then pooled in sterile disposable bags for up to 20 hours while carrying out an in-process test (holding step I). Selected fractions need to be combined to achieve the desired isoform distribution standard. To determine which fractions are pooled, a small sample of the fractions is prepared and analyzed by capillary zone electrophoresis (CZE). The fraction combination that most closely approximates the desired isoform distribution profile is preferably selected and these fractions are pooled for further purification.

  During use, 20 mM Tris. The resin is regenerated by rinsing with HCl, 1M NaCl, pH 7.0, followed by washing with WFI. The column is then sterilized with 1M NaOH for 1 hour and washed with WFI. Store the column in 0.01 M NaOH.

  Further information on chromatography resins is provided in the manufacturer's Regulatory Support File (Representative GE Healthcare Q-Sepharose Regulatory Support File).

EPO molecule unglycosylated at Ser126 residue and removal of contaminants by RP-HPLC using C4 resin RP-HPLC using C4 resin separates EPO from potential contaminants based on hydrophobicity, and If desired, the amount of EPO molecules that are not glycosylated at the Ser126 residue can be reduced. In addition, this step removes residual dye ligand that may have leached from the first column. The filtered anion exchange chromatography fraction pool is purified by reverse phase chromatography as specified in Table 5.

ＲＰ−ＨＰＬＣの性能についてのパラメータ

Parameters for RP-HPLC performance

  The column is packed with C4 resin. The column is equilibrated with 0.1% (v / v) TFA in WFI. Prior to use, column performance is defined by determining plate number and asymmetry factor. The sample is loaded onto the column and then eluted using the linear gradient described in Tables 1 and 6. The eluate is collected when the absorption at 280 nm reaches 0.01 AU and stopped when the absorption decreases to 40% to 45% of the maximum peak on the negative gradient. Collect the eluate in a glass bottle.

  As described, the gradient is formed by water / TFA 0.1% (solvent A) and water 30% / acetonitrile 70% (v / v) / TFA 0.1% (solvent B). In particular, a pool of thawed fractions of Q Sepharose HP eluate is used as a sample and filtered through a 0.2 μm filter. After rinsing the column with 2 column volumes (CV) of Milli-Q water and equilibrating with 4 CV of solvent A, the sample is loaded onto the column and the gradient is applied according to Table 6.

ＲＰ−ＨＰＬＣの性能についての勾配

Gradient for RP-HPLC performance

  The eluate is collected in 50 ml fractions and stored at −20 ° C.

During use, the column is calibrated by regenerating the resin and measuring the theoretical plate and the asymmetry factor. First, the column is washed using a gradient from 70% (v / v) acetonitrile to 100% (v / v) acetonitrile. The column is then washed with 100% (v / v) acetonitrile. The acetonitrile concentration is reduced by washing with a gradient from 100% (v / v) acetonitrile to 60% (v / v) acetonitrile, and the column is then stored in 60% (v / v) acetonitrile.

  Further information on chromatographic resins is provided in the manufacturer's Regulatory Support File (Representative Vydac C4 Resin Regulatory Support File).

Virus inactivation by incubation of EPO in acetonitrile / TFA After the RP-HPLC step, the eluate is kept at 22 ± 3 ° C. for 60-180 minutes. The acetonitrile concentration in the eluate is about 41% (v / v) and the TFA concentration is about 0.1% (v / v). During this phase of the process, the holding temperature and holding time are controlled.

Removal of RP-HPLC solvent and aggregated EPO species by cation exchange chromatography using MacroPrep High S Cation exchange chromatography using MacroPrep High S resin is used to remove RP-HPLC solvent and aggregated species and buffer Change fluids in a fairly short time. As specified in Table 7, the RP-HPLC eluate is processed by cation exchange chromatography followed by 0.2 μm filtration of the MacroPrep eluate.

カチオン交換クロマトグラフィーを実施するためのパラメータ

Parameters for performing cation exchange chromatography

  The column is packed with MacroPrep High S resin. After packing, the column is tested for theoretical plates and asymmetry factors. The column is rinsed with WFI and then sterilized with 1M NaOH for at least 1 hour. After sterilization, the column is rinsed with WFI. Prior to loading, the column was loaded with 100 mM glycine. Pre-equilibrate with HCl, pH 2.0, then equilibrate with 20 mM glycine-HCl, pH 2.0. The sample is loaded onto the column and the column is washed with 20 mM glycine-HCl, pH 2.0. The sample is eluted using the steep slope described in Table 1. The eluate is collected when the absorption at 280 nm reaches 0.03 AU and stopped when the absorption reaches 0.03 AU. Store the eluate in a sterile disposable bag. The cation exchange chromatography eluate is filtered using a 0.2 μm filter unit before further purification and / or before starting a hold time of up to 20 hours in a sterile disposable bag at 22 ± 3 ° C.

Recycle the resin during use. The column is washed with 10 mM NaPO ₄ , 1.0 M NaCl, pH 7.2, and then rinsed with WFI. EPO aggregates elute in the regenerated product. Sterilize the column with 1M NaOH for at least 1 hour and rinse with WFI. Store the column in 0.01 M NaOH.

  Further information on the resin is provided in the manufacturer's Regulatory Support File (Representative BioRad MacroPrep High S Regulatory Support File).

Ultrafiltration Optionally, the filtered cation exchange chromatography eluate is further concentrated by ultrafiltration using a 10 kDa cutoff tangential flow filtration unit to achieve the desired volume reduction. Information regarding the filtration step is listed in Table 8.

限外ろ過を実施するためのパラメータ

Parameters for performing ultrafiltration

The filter unit is sterilized with 1M NaOH and then washed with WFI. After washing, the filter unit is equilibrated with 10 mM NaPO ₄ , 0.15 M NaCl, pH 7.2 buffer. The sample is then loaded and then filtered at 22 ± 3 ° C. with a feed pressure of 0.7-1.1 bar. Collect the remainder in a sterile disposable bag.

Removal of any residual EPO aggregates and other contaminants by size exclusion chromatography using Superdex S200 Prep Grade Superdex S200 prep grade size exclusion chromatography is a final (polishing) chromatography step that can be used to Remove any agglomerates that may be present and formulate the bulk drug substance solution. The concentrate after ultrafiltration (Example 7) or the eluate after CEX chromatography (Example 6) was processed by size exclusion chromatography as specified in Table 9, followed by the description in Example 9. Then, 0.2 μm of the SE-HPLC eluate is filtered.

サイズ排除クロマトグラフィーを実施するためのパラメータ

Parameters for performing size exclusion chromatography

The column is packed with Superdex S200 prep grade resin. After packing, the column is tested for theoretical plates and asymmetry factors. The column is sterilized with 0.5 M NaOH for 1 hour and then rinsed with WFI. Prior to loading, the column is pre-equilibrated with 100 mM NaPO ₄ , pH 7.2, and then equilibrated with 10 mM NaPO ₄ , 0.15 M NaCl, pH 7.2. The sample is loaded onto the column, the eluate is collected when the absorption at 280 nm increases above 0.01 AU, and stopped when the absorption reaches 0.01 AU. Collect the eluate in a sterile disposable bag. The size exclusion chromatography eluate is filtered through a 0.2 μm filter into a sterile disposable bag prior to further processing.

  After use, the column is washed with WFI. The column is then sterilized with 0.5-1 M NaOH (0.6 CV) for 1 hour and stored in 0.01 M NaOH.

  Further information on the resin is provided in the manufacturer's Regulatory Support File (Representative GE Healthcare Superdex Regulatory Support File).

Virus removal from EPO preparations by nanofiltration Finally, a 15 nm nanofiltration step is included in the purification process to increase the virus safety of the drug substance. The size exclusion chromatography elution filtrate is nanofiltered using a Planova 15N filter that serves to remove viral exogenous factors from the eluate. The nanofilter unit is prepared by flushing with 150 ml of 10 mM NaPO ₄ , 0.15 M NaCl, pH 7.2. The 0.2 μm filtered size exclusion chromatography eluate is pumped through the nanofilter and the filtrate is pooled in a sterile disposable bag.

Filling, Storage and Transportation The filling process is the final step in the EPO drug substance manufacturing process. The final container is sterilized and filled after removing the pyrogen. The nanofiltered fraction is dispensed into a bulk drug substance storage container with a volume of 30 ml using a peristaltic pump. This operation is carried out in a laminar flow hood (local protection) in a filling chamber at ambient temperature. Storage of the final product can be done at 70 ° C., for example in a Teflon-coated PP tube. The drug substance can be transferred to the drug product manufacturing site and the bulk drug substance temperature is maintained below -70 ° C. with dry ice during transport.

Claims (22)

A method of purifying a bioactive erythropoietin (EPO) isoform O- glycosylated at a Ser126 residue from a complex protein mixture in a pure form suitable as a drug substance, comprising the following steps:
(A) Dye affinity chromatography to capture, concentrate and provide a significant reduction in potential contaminants for EPO-containing solutions;
(B) Anion exchange chromatography for concentration of acidic isoforms of EPO, further removal of contaminants, and removal of dye ligands that may have leached from the first column;
(C) Reversed phase high pressure liquid chromatography (RP-HPLC) under conditions to remove EPO molecules that are not glycosylated at Ser126 residue and to remove contaminants;
(D) cation exchange chromatography to remove RP-HPLC solvents and aggregated species, exchange buffers, and concentrate EPO fractions; and
(E) Size exclusion chromatography as a final chromatography step used to remove any potentially remaining aggregates and other contaminants;
Are done in this order . The method of claim 1, wherein elution of EPO in one or more chromatography steps is performed by step or gradient elution. Anion exchange chromatography steps can be performed using diethylaminoethyl group (DEAE), quaternary aminoethyl group (QAE), quaternary ammonium group (Q), dimethylaminoethyl group (DMAE) and / or trimethylaminoethyl group (TMAE). It carried out using an anion exchange resin or membrane containing a functional group, a method according to claim 1 or 2. 4. The method of claim 3 , wherein the anion exchange chromatography step is performed using commercially available Q-Sepharose. The method according to any one of claims 1 to 4, wherein RP-HPLC is carried out using a resin containing a methyl group, a butyl group, a phenyl group, a propyl group and / or an octyl group as a functional group. 6. The method of claim 5 , wherein RP-HPLC is performed using commercially available C4 reverse phase chromatography material. 7. The method according to any one of claims 1 to 6 , wherein the cation exchange chromatography step is carried out using a resin comprising a sulfopropyl cation exchange material. 8. The method of claim 7 , wherein the cation exchange chromatography step is performed using a commercially available Macro-Prep High S. The method according to any one of claims 1 to 8 , wherein the affinity chromatography step is carried out using a dye chromatography resin. 10. The method of claim 9 , wherein the affinity chromatography step is performed using commercially available Blue-Sepharose. Size exclusion chromatography step, Superdex, Sephacryl, Sephadex, Sepharose , Fractogel, Toyopearl, and Bio-Gel performed using gel filtration medium is selected from the group, according to any one of claims 1 to 10 the method of. 12. The method of claim 11 , wherein the size exclusion chromatography step is performed using a commercially available Superdex-S200. 7 . 13. The method according to any one of claims 1 to 12 , wherein a linear salt gradient of 0-200 mM NaCl in a buffer containing 0 mM 20 mM Tris-HCl is used. 14. The method according to any one of claims 1 to 13 , wherein EPO is eluted with a linear gradient of organic solvent in the RP-HPLC step . A linear gradient of 0-70% acetonitrile in water was used, and the solvent was 0 . 15. The method of claim 14 , comprising 1% TFA. Acetonitrile and 0. 16. The method of claim 15 , wherein isocratic elution of EPO with a solvent containing 1% TFA in water is used. Before one or more chromatography steps, ultrafiltration step, and optionally including a nanofiltration step as the final step by the method according to any one of claims 1 to 16. Following steps
18. The method according to any one of claims 1 to 17 , further comprising ( f) a nanofiltration step; and (g) an ultrafiltration step prior to steps (a), (b) and / or (e). . The method according to any one of claims 1 to 18 , wherein the EPO is human recombinant EPO. 20. The method according to any one of claims 1 to 19 , wherein the EPO is human recombinant EPO produced in CHO cells. 21. A pharmaceutical composition comprising a preparation of a bioactive EPO isoform O- glycosylated with a Ser126 residue purified by the method of any of claims 1 to 20 , wherein the preparation comprises less than 1% A pharmaceutical composition comprising a non-O-glycosylated EPO isoform . The pharmaceutical composition according to claim 21 , wherein the EPO is human recombinant EPO.

Images